HRMS-Targeted-DIA methodology for quantification of wastewater-borne pollutants in surface water

A robust method was developed for the quantification of popular and highly occurrence contaminants of emerging concern from wastewater treatment plant effluents and is explained in detail. A homemade multi-layered and multi-sorbent solid phase extraction (SPE) cartridge was used to cover the wide range of polarities of the selected contaminants. A non-discriminant elution protocol was also applied. Liquid chromatography coupled to a high-resolution mass spectrometer (HRMS) Q-Exactive Orbitrap system was used for the separation and detection of the contaminants. A targeted data independent acquisition (DIA) mode with an inclusion list with the exact mass, retention time window and collision energy was tried for the first time obtaining good sensitivity, selectivity and high quality MS2 product ions.• 116 compounds of a wide-scope of polarities and physic-chemical properties were validated using a surface water pool matrix.• SPE followed by LC—HRMS with a targeted DIA was used for the method validation at three concentration levels 5, 50, 500 μg l-1 in extract.• Good recoveries were obtained between 70 and 120% for the majority of the selected contaminants. Matrix effect, precision, and linearity were also evaluated and results proved the suitability for the method application.

a b s t r a c t A robust method was developed for the quantification of popular and highly occurrence contaminants of emerging concern from wastewater treatment plant effluents and is explained in detail. A homemade multi-layered and multi-sorbent solid phase extraction (SPE) cartridge was used to cover the wide range of polarities of the selected contaminants. A non-discriminant elution protocol was also applied. Liquid chromatography coupled to a high-resolution mass spectrometer (HRMS) Q-Exactive Orbitrap system was used for the separation and detection of the contaminants. A targeted data independent acquisition (DIA) mode with an inclusion list with the exact mass, retention time window and collision energy was tried for the first time obtaining good sensitivity, selectivity and high quality MS2 product ions.
• 116 compounds of a wide-scope of polarities and physic-chemical properties were validated using a surface water pool matrix. • SPE followed by LC -HRMS with a targeted DIA was used for the method validation at three concentration levels 5, 50, 500 g l -1 in extract. • Good recoveries were obtained between 70 and 120% for the majority of the selected contaminants. Matrix effect, precision, and linearity were also evaluated and results proved the suitability for the method application.

Specifications table
Subject area: Environmental Science More specific subject area: Environmental Analytical Chemistry -Organic Contaminants Name of your method: CECs targeted method for surface water Name and reference of original method: This analytical method is based on the method described by Enelton Fagnani, Nicola Montemurro and Sandra Pérez (2022). Multilayered solid phase extraction and ultra performance liquid chromatographic method for suspect screening of halogenated pharmaceuticals and photo-transformation products in freshwater -comparison between data-dependent and data-independent acquisition mass spectrometry , Journal of Chromatography A https://doi.org/10.1016/j.chroma.2021.462760 Resource availability: NA

Background
Water is a basic need that is necessary in our day to day life and is impossible to do without, thus its constant use. After its use, different treatments are applied to ensure its return to the ecosystem of origin (back to rivers, sea or ocean) or for its future reuse. Wastewater is usually managed through wastewater treatment plants, (WWTPs), especially in developed countries, and it may contain several contaminants such as nutrients, metals, parasites and contaminants of emerging concern (CECs) [1] . However, it has been demonstrated that WWTPs are not sufficiently efficient in their removal and many wastewater-borne pollutants are continuously being released back into the environment. Through WWTPs, wastewater-borne pollutants are introduced into surface waters, where sometimes the wastewater effluent can even dominate those streams and rivers where low flow conditions are present. Wastewaterborne pollutants enclose various types of contaminants and are of growing concern. New pollutants are continuously detected and many compounds with different physicochemical properties and different polarities are found to be detected after the WWTP discharge [ 2 , 3 ]. However, discharge guidelines and standards are not always available, especially concerning most pharmaceuticals or personal care products even though their use is continuously increasing. Therefore, a targeted method was developed to be able to track and control the continuous release of these compounds by WWTPs as they should be monitored due to their potential undesirable effect on the environment.
In this work, an extraction method based on a multi-layered and multi-sorbent solid phase extraction was combined with a nondiscriminant elution protocol to cover a wide range of different contaminants of emerging concern that end up in rivers mainly derived from WWTPs effluent. To study the method's effectiveness, the method was validated for a total of 116 compounds in surface water samples.

Chemicals and reagents
The analytes included for this targeted analysis were selected concerning their environmental occurrence as well as their presence in the list of priority substances in surface water from the EU Water Framework Directive [4] and previous group experience [5][6][7] . The list of selected compounds for a total of 116 compounds with a wide range of polarities (Log P ranging from -2.16 to 5.62) and different physicochemical properties are given in Table 1 including the name of the target compound, CAS number, molecular formula, monoisotopic mass, Log P and classification of compounds. Several classes of CECs are included in this list, mainly pharmaceuticals, UV filters, stimulants, sweeteners and some critical metabolites and transformation products (TPs). Extended details of the compounds are also included in Table SM 1. All analytical reference standards were of high purity and purchased from Sigma Aldrich (St. Louis, MO U.S). Isotopically labelled compounds used as surrogates for calibration purposes were purchased from Cerilliant (Sigma Aldrich, St. Lous, MO, U.S), Santa Cruz Biotechnology (Dallas, TX, US.), or Toronto Research Chemicals (Toronto, ON, Canada) and are listed in Table 5 in the supplementary material. For standard, samples, and calibration curve preparation, pure water, methanol (MeOH), acetonitrile (ACN) and ethyl acetate (EtOAc) of high-performance liquid chromatography (HPLC) grade were obtained from J.T. Backer (Deventer, The Netherlands). Formic acid ( ≥ 98.0%, ACS grade), ammonium acetate (AcNH 4 ), and concentrated ammonia solution (NH 4 OH) (29.0%, ACS grade) were supplied by Sigma-Aldrich. Ammonium fluoride (NH 4 F) was purchased from Fisher Chemical (Fisher Scientific SL, Madrid, Spain). The solid phase extraction (SPE) sorbents for the homemade SPE cartridges preparation were Oasis HLB, WAX, and WCX (60 μm particle size) purchased from Waters Corp., (Milford, MA, USA), while Bond Elut PPL (63-150 μm particle size) was purchased from Agilent Technologies Inc. (Santa Clara, CA, USA). Empty 6 mL polypropylene cartridges were purchased from Phenomenex Inc. (Torrance, CA, USA).

Sampling and sample pre-treatment
Surface water samples were collected from six different municipalities from Osona, a region in Catalonia, Spain. These municipalities were chosen as they have their own WWTP and a direct discharge into their respective municipality stream. For method development and validation, samples were taken from upstream points, approximately 100 m before the WWTP discharge point to avoid possible direct contamination from the discharge points and were collected in 1-L amber PET bottles, which were previously rinsed with sample water on site. The bottles were placed in a refrigerator at 4 °C under dark conditions and transported to the laboratory. Once in the laboratory, samples were immediately filtered with 0.7-μm glass microfiber filter GF/F from Whatman (UK). An aliquot was stored in 0.5 L flasks to be extracted directly or stored in the dark at -20 °C. The remaining portion was blended with all upstream points to generate a representative pool of contaminant-free surface water from the six different streams to be used for method validation and the corresponding matrix-matched curve. In Table 2 some basic water parameters are shown (pH and electrical conductivity (EC)) for each of the studied municipalities and the sample pool made.

Extraction procedure
Aliquots of 500 mL of surface water pool were extracted using a solid phase extraction. A homemade multi-sorbent SPE cartridge, previously developed by [8] , was used for the extraction. This cartridge consisted of two layers, an upper layer containing 200 mg of Oasis HLB sorbent and a bottom layer containing a mixture of 150 mg of Bond Elut PPL, 100 mg of WAX and 100 mg WCX. Activation and conditioning of the cartridges were performed by passing 2 x 5 mL of MeOH followed by 2 x 5 mL of HPLC water.    Table 3 Gradient profile, time, percentage of mobile phases and flow rate.

Instrument analysis
The targeted contaminants of emerging concern were separated using ultra high-performance liquid chromatography in a Waters Acquity HSS T3 (C18) column (100 × 2.1 mm i.d., 1.8 μm particle size) thermostated at 40 °C using a Waters ACQUITY UHPLC system (Waters, Milford, MA). For the detection, a Q-Exactive Orbitrap mass spectrometer (Thermo-Fisher Scientific, Germany) equipped with heated electrospray ionization (HESI) was used. The tuning methods and parameters used for the MS acquisition were optimized in the following conditions; spray voltage was set to 3.5 kV (positive) and 2.5 kV (negative), S-Lens RF level, 60; capillary temperature, 350 °C; auxiliary gas temperature, 300 °C. Nitrogen was used as sheath gas flow rate 40; auxiliary gas flow rate 10 and sweep gas flow rate 2. For the positive electrospray ionization (ESI + ), the mobile phases used were ACN and 5 mM AcNH 4 + 0.1% formic acid in water while for the negative electrospray ionization (ESI-) mode ACN and 2 mM NH 4 F in water were used. The gradient profile was the same for both ionizations as shown in Table 3 . The injection volume was set to 10 μL.
As for the mass spectrometer, a full-scan experiment followed by a targeted data-independent acquisition (targeted-DIA) mode were used with a specific predefined inclusion list which contained the exact mass of the selected precursor ion, optimized retention time windows and optimized collision energies in order to obtain high quality MS2 product ions (Table SM 2 and 3). For the retention time optimization, a mix of the analytes at 10 g l -1 was initially evaluated with the specific selected column by a full-scan MS with a targeted data-dependent MS2 (dd-MS2) acquisition mode in order to obtain the compound retention times and study possible coelutions. Then, the predefined inclusion list was compiled. The MS parameters for the selected method were optimized and described as follows: full MS (70,000 K resolution, AGC target 3x10 6 , IT 150 ms, scan range 90-1000 m/z ) followed by a DIA experiment to generate MS2 spectra (17,500 resolution, AGC 2x10 5 , IT auto, Loop count 1, Top N 10, isolation window 1.5 m/z , and NCE 30 eV). As mentioned above, the inclusion list was included with the exact mass to generate as many micro-scanning events as precursor ions filtered. In this way, greater sensitivity and selectivity were achieved by this method. Further information on specific details is included in the supplementary material (Table SM 1) or elsewhere [4] .
The guidance SANTE 11,312/2021 [9] was followed, and two ions were used for high-confidence peak identification criteria. For quantification, the presence of the precursor ion in the extracted chromatogram (signal-to-noise ratio > 3) with a mass accuracy within 5 ppm and matched retention time within 0.1 min compared to the reference standard was applied. For further confirmation, the presence of at least one product ion in the MS2 spectrum with a mass error below 5 ppm was expected. Only the adduct ion obtained from the full-scan spectrum was used for the isotopically labelled compounds.

Method validation
Surface water pool samples were used for the method validation, and aliquots of 500 mL were spiked with the mix of the 116 selected targeted compounds. The method was validated, in triplicate, at three different concentration levels: 5, 50, and 500 g l -1 in the extract, which would correspond to final sample concentrations of 5, 50, 500 ng l -1 respectively, after applying the method concentration factor of 1000. These concentrations were chosen in order to cover common environmental concentrations. The method was evaluated in terms of accuracy (recovery rates), matrix effect (ME), precision (% RSD), linearity (R 2 ), method detection limits (MDL), method quantification limits (MQL) according to [10] , for all three validated concentrations.  In order to evaluate the accuracy of the method developed, recovery rates (RR) were calculated, for both intra and inter day precision. The recovery rates were calculated by comparing the peak area of the pool samples spiked before and after the method extraction. A blank sample without any spiked compounds was subtracted in each case ( Eq. (1) ) The recovery rates obtained for the present method are shown in Fig. 1 . The recovery rates were generally good for most of the compounds ranging from 70 to 120% with the exception of some compounds such as cyclamate, irgasan, losartan, lidocaine or paroxetine which presented recovery values of 26, 20, 19, 38 and 19% respectively at the lowest level of validation. Nevertheless, for the high validation level (500 g l -1 in the extract) more than 80 compounds were recovered with recovery values higher than 70%. At the same time, those compounds with low recovery rates at the low validation level, are usually found at high concentrations in the environment [ 11 , 12 ] and with the use of isotopically labelled compounds these can be quantified and thus those compounds were included in the method anyway.
Matrix effect was also studied to relate either signal suppression or enhancement and was calculated for the selected compounds and for the three validation levels. Eq. (2) was applied where the area from a spiked matrix sample (post-spiked) was compared with the area given in a spiked solvent sample at the same given concentration. The area from a blank matrix (non-spiked) was subtracted.
The matrix effects for the validated compounds for each validation level are shown in Fig. 2 . Mainly ion suppression was observed in all three cases, and as the concentration increased, a slight increase or change from rather matrix enhancement to suppression was observed. There were some exceptional cases where an important enhancement was observed for hydroxychloroquine, azithromycin or carazolol.
Precision was evaluated by both intra and inter-day relative standard deviation (RSD), shown in Table 4 , to study the repeatability and reproducibility. Good results were obtained for the studied method with intra-day RSD values from 0.43 to 20.63% and inter-day values from 0.06 to 19.31%, with the exception of crotamiton and fluticasone, which gave slightly higher RSDs. MLODs and MLOQs were estimated by the standard deviation of the intercept divided by the slope, multiplied by three and ten respectively. Linearity Table 4 Relative standard deviation, method limits of detection and quantification, and linearity (R 2 ) for each compound.  The method also proved to be highly sensitive with MLODs in the range of 0.01-1.89 g l -1 and MLOQs ranging from 0.05 to 6.31 g l -1 .

Compound name Intraday RSD (%) Interday RSD (%) MLOD (ng mL -1 ) MLOQ (ng mL
Quality assurance and quality control were ensured by preparing procedural blanks using HPLC water and repeating the exposed method procedure. No peaks were observed for any of the target analytes. Quality controls were also prepared by spiking the same solvent ratio as the final extracts with standards and isotopically labelled compounds to reach a concentration of 50 g l -1 in the vial, and these were injected and analysed along the sample sequence analysis to ensure reliable determination. To overcome matrix effects, isotopically labelled compounds were used in all samples as well as a matrix-matched calibration curve. For proper quantification, the matrix-matched calibration curve included 10 points with the 116 mix standards and 80 isotopically labelled compounds.

Method applicability
The WWTP discharge upstream samples of each municipality and the surface water pool used for the method validation and matrixmatched calibration curve ( Table 2 ) were analysed as an example of method applicability and results are shown in Fig. 3 . In general, very low levels of contamination were found. The most contaminated upstream was the Vidrà municipality with a total accumulated concentration of 864.2 ng L -1 , followed by the Taradell municipality with 433.7 ng L -1 . In Fig. 3 , those contaminants present with at least 20 ng L -1 in one sample are shown. As expected, many of the selected contaminants were not detected (BP4, diclofenac, oxazepam, valsartan) as their presence is mainly expected in surface rivers after the wastewater treatment plant discharge. However, a total of 48 compounds of the 116 targeted compounds were detected in at least one of the samples. The highest concentrations found were mainly concerning those compounds used frequently in our day to day life such as sucralose (sweetener) with the highest concentration of 376.9 ng L -1 found in Taradell followed by caffeine (stimulant), acesulfame or saccharin (sweeteners) with maximum concentrations of 179.4, 130.3 and 119.0 ng L -1 respectively.
Overall, the method developed proved a very good performance for the quantification of a wide range of wastewater-borne pollutants with different polarities. Thus, it has a high applicability for monitoring and controlling the impact of wastewater-borne pollutants in surface water and to further explore possible other contamination sources other than the continuous WWTP discharges.

Declaration of Competing Interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

Data Availability
Data will be made available on request. ence and Innovation and the IDAEA-CSIC , a centre of Excellence Severo Ochoa ( CEX2018-000794-S ). The authors also acknowledge the support from the Economy and Knowledge Department of the Catalan Government through Consolidated Research Group ( ONHEALTH, 2021 SGR 01150 ). The EU is not liable for any use that may be made of the information contained therein. NM thanks the Grant RYC2021-031725 -I funded by MCIN/AEI/ 10.13039/501100011033 and, as appropriate, by "ESF Investing in your future "

Supplementary materials
Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.mex.2023.102093 .